GCT is a specific type of CNS tumor with several subtypes. The two major forms of these tumors, germinoma (GPG) and NGMGCT (IPG/PPG), present with different clinical behaviors, differences in sensitivity to therapeutic regimens and different outcomes. The overall survival of patients with germinomas is significantly better than that of patients with NGMGCTs in our series (Figure ) and this is similar to other previously reported series [42
]. To explore the molecular difference between these two different histological/therapeutic prognostic groups, we have identified with confidence a number of differentially expressed miRNAs and mRNA; these permit an interpretation of the clinical survival variations and downstream hypothesis testing. The various divergent biological functions that correlate with the clinical observations are also revealed.
Among these miRNAs, miR-142-5p and miR-146a are upregulated in the pediatric germinomas (GP group) when compared to the NGMGCTs (IPG/PPG). Up to the present, no miRNA profile of pediatric GCTs has been published. A miRNome report on adult gonadal GCTs showed that, for each GCT subtype, the miRNA patterns are quite different [24
]. In their dataset, miR-142-5p and miR-146a are also more abundant in adult seminomas than in gonadal ECs [24
]. In addition, let-7e, miR-133b, miR-218 and miR-654-3p are also abundant in both pediatric NGMGCTs and adult ECs (Figure ) [24
]. However, the notable discrepancies are miR-181c and miR-218, the expression levels of which are more abundant in adult testicular seminomas but are lower in pediatric intracranial germinomas (Figure ) [24
]. The unique expression pattern of these miRNAs in pediatric CNS GCTs may reflect the differences in pathogenesis mechanisms between adult and pediatric GCTs [17
], or, alternatively, the variation in genetic makeup between Western and Taiwanese patients.
We also correlated the transcript levels of miRNAs to their candidate targets in order to identify microRNA-mRNA target pairs (Table ). It has been shown that some miRNAs, such as miR-1, can downregulate the transcript levels of a large number of target genes in mammalian cells [18
]. Two large scale proteomic studies published very recently have shown that, although some microRNA target proteins are repressed without detectable changes in mRNA levels, more than a third of translational repressed targets also display detectable mRNA destabilization and, for the more highly repressed targets, mRNA destabilization usually makes up the major component of repression [19
]. Gene expression microarrays can therefore be, and have been, applied for the identification of downstream targets for miRNAs [44
]. However, proof of direct binding between those miRNAs and target mRNAs, as well as the direct translational repression of target mRNAs, is still needed. Such confirmation will require more wetlab experiments such as immunoblotting and reporter assays.
When compared with NGMGCTs, the germinomas largely recapitulate the features of self-renewing pluripotent human embryonic stem (hES) cells, such as involvement of POU5F1 (OCT4), NANOG and KLF4 (q
< 0.01). Both seminomas and embryonal carcinomas are known to express stem cell markers, such as POU5F1 and NANOG [47
]. In addition, in an attempt to find coordinated overexpressed gene clusters in GCTs, Korkola et al
. found NANOG at chromosome 12p13.31 is overexpressed in undifferentiated (embryonal carcinomas and seminomas) tumors versus differentiated (teratoma, yolk sac tumor, and choriocarcinoma) tumors [16
]. By overexpressing POU5F1, NANOG and KLF4, it is now possible to reprogram the transcriptomes of somatic primary cells, which results in their dedifferentiation from matured cells to ES cell-like iPS (induced pluripotent stem) cells [49
]. The abundant expression of these dedifferentiation factors in germinomas therefore mirrors the more undifferentiated histopathological characteristics of these tumors. Whereas such similarities have previously been described for adult and pediatric seminomas [16
], we now know that this also applies to Asian pediatric CNS germinomas.
Although germinomas abundantly express the above three stemness factors, it is NGMGCTs (IPG/PPG) who show a closer gene expression pattern to ESCs (Figure ). This observation is consistent with pervious global gene expression reports whereby the gene expression patterns of human ES cell lines are similar to those of the human embryonal carcinoma cell samples but are more distantly related to those of seminoma samples [12
]. The close relationship between NGMGCTs and ES cells supports the hypothesis that germinomas are closely related to primordial germ cells (PGCs), and EC cells/NGMGCTs represent a reversion to a more ICM- or primitive ectoderm-like cell type [12
]. Whether germinomas and zygotes/blastomeres share similar mRNA or microRNA profiles is under investigation at present. The close relationship between NGMGCTs and ES cells may additionally be reflected in the worse prognosis for these tumors. Recently, via novel genomic approaches, it has been shown that aggressive and poor prognostic tumors, such as glioblastomas, inherit preferential ES cell gene expression profiles [36
]. The similarity between pediatric NGMGCTs and human ES cells may therefore reflect the clinical observation that CNS NGMGCTs are more malignant and show a higher fatality rate than germinomas.
The close relationship in genetic makeup between NGMGCTs and ESCs also suggest that factors other than POU5F1 (OCT4), NANOG or KLF4 are responsible for ESC gene expression. In this study, we found that two key epithelial-mesenchymal transition (EMT) regulators, SNAI2 (SLUG) and TWIST2, are abundantly expressed in the NGMGCT group (IPG/PPG) (Table and Figure ). It has been reported that EMT transcription factors, SNAI1 (alias SNAIL) and TWIST, can independently dedifferentiate mammalian cancer cells and induces the generation of cancer stem-like cells, which then form mammospheres [50
]. It is possible that SNAI2 (SLUG) and TWIST2 behaves like Snail and TWIST and can introduce malignancy and stemness in pediatric GCTs. Targeting oncogenic stemness genes or EMT-related embryonic signaling pathways (such as the Wnt pathway, Figures &) may differentiate a highly malignant NGMGCT into a more matured transcriptome type, thereby increasing the sensitivity of these tumors to the classical therapeutic regimen of radical resection, irradiation and chemotherapy, which would produce a better prognosis for the patients.
In addition to stemness genes (such as genes involved in reproduction and male gonad development), the germinomas were found to overexpress genes involved in the DNA damage checkpoint, which indicates active DNA integrity checking in the germinomas and thereby reflects why the clinical phenotype of germinomas has a better prognosis (Figure ). Among the other genes that were found to be expressed abundantly in germinomatous tissues were genes associated with the immune system process and this correlates with the abundant lymphocytic infiltration of germinomas found during histological observation. Relative to germinomas, we observed a significant enrichment of overexpression of differentiation and morphogenesis (especially neurogenesis) genes in NGMGCTs, which correlates with the differentiated state of these tumor cells (Figure ). There is also evidence of overexpression of genes in the Wnt/β-catenin pathway in our dataset (Figures ), which is consistent with previous studies of nonseminomatous malignant GCTs [15
]. In concordance with the higher recurrence and disseminating clinical behaviors of NGMGCTs, a significant enrichment for overexpression of motility, tight junction, focal adhesion, and adherent junction genes in NGMGCTs was observed (Figures ). Our results thereby integrate molecular profiles with clinical observations and provide a better understanding of the underlying molecular mechanisms. The combined targeting of hub genes involved in all these biological modules by a cocktail therapy-like regimen may eventually lead to an alleviation of these malignant CNS tumors.
During the submission of this manuscript, a very recent reference based on testis GCTs identified gene expression signatures that predicted outcomes in patients with extra-cranial adult GCTs [52
]. We compared the age and tumor characteristics between our series against the genomic study group of CNS GCTs in children and the reported study of extra CNS GCTs in adult men (Additional file 1-C
]. In our series and the genomic study of CNS GCTs, both germinomas and NGGCTs in children younger than 18 years old were included, whereas Korkola's study involved adult men with nonseminomatous GCTs (NSGCTs) [52
]. In our series, 118 tumors were pure germinomas or tumors with a germinoma component, 49 tumors were pure teratoma or tumors with a teratoma component, and 27 cases were classified as YSTs including 10 pure YSTs, 11 tumors with a YST component, and 6 cases with serum AFP elevation (pure immature teratomas excluded). Among the 21 cases with genomic studies, 9 tumors were pure germinomas, 2 tumors were pure mature teratomas, and 9 tumors were mixed GCTs, including one mature teratoma with serum AFP elevation and one germinoma with serum AFP elevation. The correlation of tumor characteristics between the studies of Korkola et al
. and ours in Additional file 1-C
constituted the basis for the comparison of genomic molecular findings across the different therapeutic prognostic groups and histology between these two studies.
Korkola et al
. concluded that using a 140-gene signature, they could predict 5-year overall survival (OS) (p
< 0.001) [52
]. Both our study and that of Korkola et al
. identified good outcome GCTs express gene sets involved in immune function and the repression of differentiation (such as POU5F1/OCT4), while poor outcome GCTs express genes involved in active differentiation (in particular, neuron differentiation) (Fig. ) [52
]. A 10-gene prognosis model was also built using a univariate Cox model. When the samples were dichotomized by median score, there was significant separation of the survival curves (p
< 0.002) [52
]. These 10 genes were STX6, CFLAR, FNBP1, ITSN2, SYNE1, MAP3K5, PTGDS, PXMP2, IRAK4, and RABGAP1L [52
]. Among these 10 genes STX6 (syntaxin 6) and CFLAR (CASP8 and FADD-like apoptosis regulator) are over-expressed in our germinoma group (q
< 0.01). It will be interesting to fit their prognosis signatures onto our dataset to see whether GCTs of different anatomic locations, ages and ethnic populations express similar prognosis genes. However, since all the tissues used in our study were freshly collected over the last 2 years, only one death has been recorded so far (Additional file 1
). As a result, this work needs to be carried out at a later stage.
The variation in chromosome copy number variation (CNV) regions between germinomas and NGMGCTs were mapped to cytobands 4q13.3-4q28.3 and 9p11.2-9q13 (Figure ). Chromosome abnormality analysis of adult testicular germ cell tumors (tGCTs) revealed that all GCTs show 12p gain [25
]. In 2007, Palmer et al
. used metaphase-based comparative genomic hybridization (CGH) to analyze genomic imbalance in 34 pediatric GCTs (22 yolk sac tumors (YSTs), 11 germinomatous tumors and one metastatic embryonal carcinoma). The YSTs showed an increased frequency of 1p loss (p
= 0.003), 3p gain (p
= 0.02), 4q loss (p
= 0.07) and 6q loss (p
= 0.004) compared to germinomas [30
]. Most of their cases were from the testis, the ovary or the sacrococcygeal region and only 2 germinomas and 1 YST brain GCTs were included [30
]; this is a possible explanation of the discrepancies between their results and ours. We also observed 4q loss in the NGMGCTs (including YSTs), suggesting that genomic imbalance in this region, and the genes/miRNAs encoded by this chromosomal region, may play a crucial tumor suppressing role during NGMGCT pathogenesis and affect clinical performance (Table ). Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5) within 4q13.3-4q28.3 showed higher expression levels in the germinomas (Table ). DDIT4L, ELOVL6 and HERC5 are among the top 50 highly expressed genes in germinomas (Table ). A putative GCT tumor suppressor gene SYNPO2 (Synaptopodin 2), also known as myopodin, is also within the 4q13.3-4q28.3 deletion region (Table ). SYNPO2 has recently been shown to have the highest predictive value when assessing 5-year overall survival [52
], which is consistent with a possible role as a tumor suppressor. However, we do not observe differential SYNPO2 expression between NGMGCTs and germinomas (Table ). It is unclear whether SYNPO2 expression is also downregulated in Taiwanese germinomas compared to normal brains. In addition, whether survival predictors derived from Western cases can be applied to Asian patients still awaits elucidation.
Recently two independent genome-wide association studies (GWAS) have reported on susceptibility loci associated with tGCT: Kanetsky et al
. mapped seven markers at 12p22 near KITLG (c-KIT ligand) and two markers at 5q31.3 near SPRY4 (sprouty 4) [53
]; furthermore Rapley et al
. identified loci on chromosome 5, 6 and 12 [54
]. A third locus, in an intron of BAK1, a gene that promotes apoptosis, was also identified by Rapley et al
]. Similarly, the CGH profiles in childhood GCTs have been reported to resemble those in adults [55
]. In terms of cytogenetics differences between the different histological entities, loss of chromosome 19 and 22 material and gain of 5q14-q23, 6q21-q24 and 13q material were found to occur at a significantly lower frequency in seminoma adult tGCTs compared to non-seminoma adult tGCTs [25
]. Among Taiwanese pediatric GCTs, no common copy number variation (CNV) could be found in either the germinomas or the mature teratomas (Figure ). The divergence between our results and published Caucasian ones may be partly due to the different ethnic samples used, the application of different bioinformatics algorithms and the fact that we compared the differences between germinomas and NGMGCTs but not common aberrations across all GCTs.
In summary we have identified miRNome, mRNA signatures and CNV regions that are associated with two pediatric GCT histological entities (germinoma and NGMGCTs) and two prognostic groups (GPG and IPG/PPG). The clinical discrepancies between the two histological entities (germinomas of GPG and NGMGCTs of IPG/PPG) are therefore mirrored by their differences in global transcriptome patterns and their unique stem cell traits. One of the interesting questions that remain is whether pediatric GCTs from other ethnic background also express similar transcriptome traits and CNV regions. If Caucasian and Taiwanese GCTs possess unique transcriptome traits, therapeutic and diagnostic experience from Western countries may not be applicable directly to Asian or Taiwanese patients. Therefore, the genes and miRNAs identified here hold the potential of being novel therapeutic targets and may be used for further differentiation therapy. The Wnt pathway, for example, is activated in NGMGCTs (Figure ), and drugs targeting this specific pathway may hold potential as a treatment approach to NGMGCTs. Transdifferentiating ESC-like NGMGCTs into a benign status may also be a novel and useful tactic against these fatal pediatric tumors.